Structure of GDAP1 bound to a product of lipid peroxidation

总结。 GDAP1基因突变与周围神经病Charcot-Marie-Tooth病有关。夏科特-马- RIE-Tooth是最常见的遗传性神经疾病之一，估计在美国有12.6万人受到影响 单单是美国。GDAP1基因敲除、过度表达以及CMT的多个模型显示变化 与细胞对氧化应激反应的失调相一致。这些变化包括NRF2驱动的变化 转录活性、谷胱甘肽耗竭、氧化还原失衡和线粒体去极化。在… 同时，线粒体网络对氧化应激反应的关键方面与关键方面非常相似 CMT表型的特征：碎裂、融合缺陷和细胞内位置的改变。最后，GDAP1 是谷胱甘肽S转移酶超家族的成员，它保护细胞免受过氧化脂质的伤害 外源生物是在氧化应激条件下积累的有毒分子。这个机制不够充分- ING GDAP1的S在氧化应激反应中的作用尚不清楚。我们最近发现GDAP1可以结合 4-羟基壬醛(4HNE)，一种有毒的脂质，主要由过氧化的脂质分解形成，主要是在 线粒体。这项提议将解决两个主要问题：GDAP1能否利用4HNE作为基质，在 与典型的GST酶类似，还是在氧化应激重新启动过程中起到非酶作用？ 海绵？其次，是否存在与4HNE结合相关的构象变化或4HNE结合的结果？我们 将通过1)生化测量与4HNE中草药相关的酶参数来解决这些问题。 GST活性；2)生化确定假定活性部位口袋内重要残基的影响 4HNE结合亲和力和GST酶活性的研究3)GDAP1-4HNE络合物的结构测定 使用X射线结晶学。这些数据将定义GDAP1识别4HNE的分子机制 以促进结合并揭示与4HNE结合相关的蛋白质的构象变化。如果 GDAP1是一种酶，它将揭示它是如何促进谷胱甘肽催化4HNE的。如果GDAP1正在播放 非酶作用，由4HNE引起的构象变化将在GDAP1的功能和将发挥关键作用 在这个结构中被揭示出来。总体而言，这些研究将对塑造未来的生化和细胞基础至关重要 研究主要集中在GDAP1功能上。此外，该结构将提供所需的基础，以组成- 国家预测小分子结合伙伴，这是调节GDAP1功能的关键工具，将使 更深入地询问CMT疾病模型和迈向治疗干预的第一步。

Summary. Mutations in GDAP1 are associated with the peripheral neuropathy Charcot-Marie-Tooth disease. Charcot-Ma- rie-Tooth is one of most common inherited neurological disorders, estimated to affect 126,000 people in the United States alone. GDAP1 knockdown, overexpression, and as well as multiple models of CMT show changes consistent with dysregulation of the cellular response to oxidative stress. These include changes in NRF2-driven transcriptional activity, evidence of glutathione depletion, redox disbalance, and mitochondrial depolarization. At the same time key aspects of the mitochondrial network’s response to oxidative stress are very similar to key aspects of CMT phenotypes: fragmentation, fusion deficits and change in position inside the cell. Finally, GDAP1 is a member of the Glutathione S-transferase (GST) superfamily which protect the cell against peroxidated lipids and xenobiotics, toxic molecules that accumulate under conditions of oxidative stress. The mechanism underly- ing GDAP1’s role in oxidative stress response is unknown. We have recently discovered that GDAP1 can bind 4-hydroxynoneal (4HNE), a toxic lipid that is formed from the breakdown of peroxidated lipids primarily in the mitochondria. This proposal will address two main questions: can GDAP1 utilize 4HNE as a substrate, in a manner similar to canonical GST enzymes, or does it have a non-enzymatic role in the oxidative stress re- sponse? Secondly, are there conformational changes associated with or a consequence of 4HNE binding? We will address these questions by 1) biochemically measuring enzymatic parameters associated with 4HNE medi- ated GST activity; 2) biochemically defining the impact of important residues within the putative active site pocket on 4HNE binding affinity and GST enzymatic activity; 3) determining the structure of the GDAP1-4HNE complex using x-ray crystallography. These data will define the molecular mechanism by which GDAP1 recognizes 4HNE to facilitate binding and reveal and conformational changes in protein that are associated with 4HNE binding. If GDAP1 is an enzyme it will reveal how it facilitates catalysis of 4HNE with glutathione. If GDAP1 is playing a non-enzymatic role, conformational changes resulting from 4HNE will play a key role in GDAP1 function and will be revealed in this structure. Overall, these studies will be critical in shaping future biochemical and cell-based investigations centered on GDAP1 function. Further, the structure will provide the foundation needed to compu- tationally predict small molecule binding partners, critical tools for modulating GDAP1 function that will allow deeper interrogation of CMT disease models and a first step towards therapeutic intervention.